It is estimated that up to 5% of infants born to HIV seropositive mothers are HIV infected due to mother-to-child-transmission (MTCT). In 2005 alone, about 2.3 million infants were infected with HIV and about 2 million of these were in sub-Saharan Africa. Mortality rates are very high in infants infected with HIV. In HIV infected infants, mortality rate is as high as 26-45% by the first year of birth and 35-59% by the second, stressing the urgent need for early and accurate diagnosis and therapy. Early diagnosis of HIV could result in timely treatment of HIV infected infants and may result in lower mortality rates.
HIV DNA PCR detects close to 100% of all infections at 6-weeks of age. Roche AMPLICOR® HIV-1 DNA test version 1.5 is the most widely used commercial DNA PCR assay that is capable of detecting all subtypes of HIV and has been used for diagnosis of HIV in infants. Nucleic Acid Sequence Based Amplification (NASBA) has been shown to be as sensitive and specific as DNA PCR. Recently, dried blood spots (DBS) based DNA PCR has been developed for early diagnosis of HIV in infants. It does not involve venipuncture and only requires a sterile lancet for a heel prick. Briefly, a blood sample is collected on 903 filter paper (Whatman Inc., Kent, UK) and dried. Blood can be stored and transported easily in the form of DBS. Furthermore, it has been demonstrated that there is a 100% correlation between diagnosis from heel prick blood and venous blood by DNA PCR. Many groups have reported successful PCR with high sensitivity and specificity from DNA extracted from DBS. DBS have resulted in improved accessibility to rural areas. A DBS sample is collected from infants at the POC when they are brought in for their first immunization at the age of 6 weeks. The samples are shipped to a central laboratory for testing and the results are then shipped back to the rural clinic in time for the second immunization at 10 weeks post-birth.
However, very often, the results do not get back in time for the second immunization resulting in high lost-to-lab and lost to follow-up rates up to 50%. Furthermore, the extraction of DNA in a central lab requires expensive equipment and trained personnel making the test inaccessible to developing nations due to cost at about 50 USD/test. In contrast, results from early POC testing can be provided to the infant's caregiver at the same appointment, and appropriate treatment can be initiated significantly reducing lost-to-follow up rates.
Many challenges must be overcome when conducting HIV DNA tests both in centralized laboratories and out in the field. Large laboratories use automated or semi-automated robotic systems for high-volume HIV viral load assays. However, sample processing is typically the most troublesome part of these tests. Currently, sample-processing procedures involve many steps, often requiring centrifugation and extraction steps. Also, these methods often do not adequately purify the target nucleic acid. They often leave inhibitory or interfering substances in the reaction mixture that can cause inhibition of the amplification reaction and result in false-negative results. The manual nature of current sample-processing techniques also can lead to specimen cross-contamination, which can cause false-positive results.
Considerable effort has been made in trying to automate the sample preparation process, since this would allow for the more widespread use of PCR or other nucleic analysis techniques. However, existing automated high-throughput systems perform multiple extraction and purification steps, and still require certain manual preparations, including sample and reagent loading, and waste removal. Hence, highly trained technicians are required to conduct the assay and maintain the instrument. The automated systems are very expensive because they use complex robotic arms to move solutions or magnetic particles and precision instruments to pipette liquids. The cost of an automated system is often difficult to justify for smaller laboratories, especially those in resource limited settings. Cross-contamination is also a problem since they employ amplification technologies. Clinical laboratories often use separate rooms for reagent preparation, sample preparation, amplification, and post-amplification analysis. For these reasons, despite the automation, DNA proviral testing is considered high-complexity tests under the Clinical Laboratory Improvement Amendments (CLIA). To date, no Nucleic Acid Test (NAT) system has qualified for CLIA-waived status, largely because of the difficulties in automating sample preparation and reagent handling.
Performing field-use or near-patient NATs involves even more challenges, especially since they will inevitably be conducted by less-experienced users in non-laboratory environments.
What is needed are fast, inexpensive, and efficient methods of preparing samples for nucleic acid analysis.